Group of nanobioengineering
The group is aimed at the bioengineering of recombinant proteins and polypeptides, developing the procedures for their purification and renaturation, studies on the properties of recombinant molecules using physico-chemical methods, as well as the approaches and methods of nanotechnology.
The group collaborates with the departments of the IBCh RAS (Laboratory of optical microscopy and spectroscopy of biomolecules, the Molecular Instruments for Neurobiology Group and the Laboratory of molecular toxinology) and with Department of Bioengineering of the Biological Faculty of the Lomonosov Moscow State University and the Laboratory of Physicochemical Foundations of Reception of the Emanuel IBHF RAS.
Bioengineering group was formed in 2010 as a part of Bioengineering department of IBCh RAS.
- Membrane engineering in living cells – bioengineering of recombinant receptor molecules that are targeted to and inserted into bacterial membrane, study of their ligand-binding activity at the surface of the cell membrane using fluorescent methods of detection.
- Development of bioengineering approaches to the production of recombinant membrane photosensitive proteins in order to study their structural organization and photochemical properties.
- Development of bioengineering methods for obtaining soluble protein and peptide ligands in the functionally active form and studying their interaction with receptor proteins.
1. A bioengineering test system was developed to study interactions of potassium channels with ligands. The test system is based on the use of fluorescently-labeled peptide probe and hybrid potassium channels that are embedded in the membrane of whole bacterial cells. The detection of ligand-receptor interactions is carried out by the method of laser scanning confocal microscopy (LSCM). Using the test system new blockers originating from animal venoms were identified that target Kv1.1 and Kv1.3 – potassium channels of important biomedical significance. With the help of molecular modeling methods, molecular determinants of interaction of peptide toxins with potassium channels were studied, and the mutant forms of peptide toxins were constructed with increased selectivity for the target channel. The principle of engineering of genetically encoded fluorescent ligands of potassium channels was developed with the aim to use them as fluorescent probes in binding studies and also for visualization of potassium channels in cells and tissues. The work is carried out in collaboration with departments of IBCh - Laboratory of Optical Microscopy and Spectroscopy of Biomolecules, Laboratory of Molecular Instruments for Neurobiology, Laboratory of Molecular Toxinology, as well as Department of Bioengineering of the Biological Faculty of the Lomonosov Moscow State University.
2. Bioengineering method was worked out for over-production of recombinant bacteriorhodopsin (Halobacterium salinarum) in E.coli expression system. Photochemical properties of recombinant bacteriorhodopsin were found to be similar to those of the monomeric form of the native protein. Recombinant bacteriorhodopsin and its mutants are used to study primary events of photocycle and mechanisms of energy transfer using the method of Femtosecond Transient Absorption Spectroscopy (in collaboration with the Laboratory of Physico-Chemical Basis of Reception, N.M. Emanuel Institute of Biochemical Physics RAS).
3. New effective bioengineering methods have been developed for producing functionally active recombinant ligands, such as: disulfide-rich peptide toxins from scorpion venoms; efrin A1 - the ligand of Ephrine receptors. The obtained peptides and proteins are used in various studies on ligand-receptor interactions.
|Oksana Nekrasova, Ph.D.||firstname.lastname@example.org, |
|Kseniya Kudryashova, Ph.D.||r. email@example.com|
|Elena Krjukova||r. firstname.lastname@example.org, |
|Sergej Yakimov||res. eng.|
|Klara Birikh, Ph.D.||sen. eng.|
|Roman Tikhonov, Ph.D.||s. r. f.|
MeKTx11-1, Kv1.2 channel –specific peptide blocker from the M.eupeus scorpion venom: structural basis of selectivity (2018-11-27)
Оksana V. Nekrasova, K.S.Kudryashova (Group of nanobioengineering, Bioengineering department), A.A. Vassilevski, A.I. Kuzmenkov, A.M. Gigolaev (Laboratory of molecular instruments for neurobiology), A.O. Chugunov, V.M. Tabakmakher, R.G. Efremov (Group of in silico analysis of membrane proteins structure, Laboratory of biomolecular modeling), A.V. Feofanov (Laboratory of optical microscopy and spectroscopy of biomolecules).
A unique high-affinity and highly selective peptide blocker of Kv1.2 channel, MeKTx11-1, from the scorpion venom Mesobuthus eupeus was studied. Peptide MeKTx11-1 and its mutant forms were produced in a recombinant form, and their receptor-binding activity was studied against a panel of Kv1-channels. Molecular modeling of interaction of these peptides with Kv1.2 channel was carried out, and key structural elements of the interactions were determined. Peptide MeKTx11-1 may be used as a novel efficient molecular tool in neurobiology to identify and study the activity of Kv1.2 channel in the presence of different isoforms of Kv1-channels.
In collaboration with S.Peigneur and J.Tytgat fromUniversity of Leuven, Belgium and A.F. Fradkov from Evrogen JSC.
- (2018). K1.2 channel-specific blocker from Mesobuthus eupeus scorpion venom: Structural basis of selectivity. Neuropharmacology 143, 228–238
An efficient method for production of recombinant α-КТх peptides – the blockers of potassium channels (2017-11-28)
A bioengineering method for production of peptide blockers of potassium Kv1 channels has been developed that provides: high yield of the target peptides (12-22 mg/l culture); retaining the native amino acid sequence of α-КТх peptides; high yield of the renatured form of the peptides with correctly formed three and four disulfide bonds; simple and reliable procedure of peptide isolation and purification. The recombinant peptides of the α-KTx family obtained by this method have the activity of the natural blockers. High affinity potassium channel blockers from scorpion venom are widely used to study the structure and function of the channels and have a promising medical value.
- (2017). Recombinant scorpion toxins: Focus on four-disulfide peptide blockers of Kv1-channels. Bioengineered 9 (1), 25–29
- (2017). Straightforward approach to produce recombinant scorpion toxins—Pore blockers of potassium channels. J Biotechnol 241, 127–135
Development of integrated transcriptomic and proteomic approach to search for blockers of potassium channels in animal venoms (2016-03-27)
Kuzmenkov A.I. , Vassilevski A.A., Grishin Eu.V.
Department of molecular neurobiology
Kudryashova K.S., Nekrasova O.V., Kirpichnikov M.P.
Laboratory of optical microscopy and spectroscopy of biomolecules
An original approach was developed to search for new ligands of potassium channels. It combines the bioengineering cellular test system and transcriptomic and proteomic analysis of animal venoms. Using this approach eight high-affinity peptide blockers of voltage-gated potassium channel Kv1.1 (including five new peptides) were found in the venom of the scorpion Mesobuthus eupeus. The proposed approach is a versatile and effective tool for directed search for blockers of potassium channels in natural venoms.
- (2015). Variability of potassium channel blockers in Mesobuthus eupeus scorpion venom with focus on Kv1.1: An integrated transcriptomic and proteomic study. J Biol Chem 290 (19), 12195–12209